The present invention relates to influenza vaccines, and particularly to peptide-based vaccines comprising conserved epitopes of both B and T-lymphocytes recognized by the prevalent HLA""s in humans.
ABBREVIATIONS: Ab: Antibodies; CTL: Cytotoxic T-lymphocytes; EID: Egg-infective dose; HA: Hemagglutinin; HAU: Hemagglutination unit; i.n.: intranasal; i.p.: intraperitoneal; NP: Nucleoprotein; PMBC: Peripheral blood mononuclear cells; TT: Tetanus toxoid.
Influenza is a public health concern, it results in economic burden, morbidity and even mortality. Influenza infection may result in a variety of disease states, ranging from sub-clinical infection through a mild upper respiratory infection and tracheobronchitis to a severe occasionally lethal viral pneumonia. The reasons for this wide spectrum of severity are explained by the site of infection and the immune status of the host. The most important characteristic of influenza, from the immunological point of view, is the rapid, unpredictable changes of the surface glycoproteins, haemagglutinin and neuraminidase, referred to as antigenic shifts and drifts. These changes lead eventually to the emergence of new influenza strains, that enable the virus to escape the immune system and are the cause for almost annual epidemics (Laver et al., 1980 and 1980a; Webster, 1982).
Immunization towards influenza virus is limited by this marked antigenic variation of the virus and by the restriction of the infection to the respiratory mucous membranes. The influenza vaccines currently available and licensed are based either on whole inactive virus, or on viral surface glycoproteins. These influenza vaccines fail to induce complete, long-term and cross-strain immunity.
Influenza virus comprises two surface antigens: neuraminidase (NA) and hemagglutinin (HA), which undergo gradual changes (shifts and drifts), leading to the high antigenic variations in influenza. HA is a strong immunogen and is the most significant antigen in defining the serological specificity of the different virus strains. The HA molecule (75-80 kD) comprises a plurality of antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are common to many HA molecules (common determinants).
U.S. Pat. No. 4,474,757 describes a synthetic vaccine against a plurality of different influenza virus comprising a suitable macromolecular carrier having attached thereto a peptide being an antigenic fragment of HA which is common to a plurality of different influenza virus strains. One of the described common determinants is the HA epitope 91-108 which is conserved in all H3 influenza subtype strains.
The nucleoprotein (NP) is located in the viral core and is one of the group specific antigens which distinguishes between influenza A, B and C viruses. In contrast to the HA, the NP is one of the most conserved viral proteins, being 94% conserved in all influenza A viruses. Influenza A virus NP-specific antibody has no virus neutralizing activity, but NP is an important target for cytotoxic T lymphocytes (CTL) which are cross-reactive with all type A viruses (Townsend and Skehel, 1984). CTL recognize short synthetic peptides corresponding to linear regions of the influenza NP molecule (Townsend et al., 1985 and 1986).
PCT International Publication WO 93/20846 describes a synthetic recombinant vaccine against a plurality of different influenza virus strains comprising at least one chimeric protein comprising the amino acid sequence of flagellin and at least one amino acid sequence of an epitope of influenza virus HA or NP, or an aggregate of said chimeric protein. Following this approach, a synthetic recombinant anti-influenza vaccine based on three epitopes was found to be highly efficient in mice. This vaccine included HA 91-108, a B cell epitope from the HA which is conserved in all H3 strains and elicits anti-influenza neutralizing antibodies, together with a T-helper and CTL epitopes from the NP(NP 55-69 and NP 147-158, respectively), which induce MHC-restricted immune responses. Each of these epitopes was expressed in the flagellin of Salmonella vaccine strain. The isolated flagella were administered intranasally to mice, resulting in protection against viral infection (Levi and Arnon, 1996).
According to the present invention, influenza peptide epitopes reactive with human cells were expressed in Salmonella flagellin and tested for efficacy in a human/mouse radiation chimera in which human PBMC were functionally engrafted. Clearance of the virus after challenge and resistance to lethal infection was found only in the vaccinated mice and production of virus specific human antibodies was also higher in this group. FACS analysis showed that most human cells in the transplanted mice were CD8+ and CD4+, indicating that the protection was mediated mainly by the cellular immune response.
The present invention thus relates to a human synthetic peptide-based influenza vaccine for intranasal administration comprising a mixture of flagella containing at least four epitopes of influenza virus each expressed individually in Salmonella flagellin, said influenza virus epitopes being reactive with human cells and being selected from the group consisting of: (i) one B-cell hemagglutinin (HA) epitope; (ii) one T-helper hemagglutinin (HA) or nucleoprotein (NP) epitope that can bind to many HLA molecules; and (iii) at least two cytotoxic lymphocyte (CTL) nucleoprotein(NP) or matrix protein (M) epitopes that are restricted to the most prevalent HLA molecules in different human populations.
The preferred B-cell HA epitope is the influenza virus hemagglutinin epitope 91-108 [HA 91-108] of the sequence:
Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu
The preferred T-helper epitopes are the influenza virus hemagglutinin epitope 307-319 [HA 307-319] of the sequence:
Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr
and the HA epitope 306-324 [HA 306-324] of the sequence:
Cys-Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr-Gly-Met-Arg-Asn-Val
The cytotoxic T-lymphocyte (CTL) epitopes used in the vaccine of the invention will change according to the population type, namely Caucasian or non-Caucasian (of Asian or African origin). For Caucasian populations, the preferred CTL epitopes are the influenza virus nucleoprotein (NP) epitope 335-350 [NP 335-350] of the sequence:
Ser-Ala-Ala-Phe-Glu-Asp-Leu-Arg-Val-Leu-Ser-Phe-Ile-Arg-Gly-Tyr
and the NP epitope 380-393 [NP 380-393] of the sequence:
Glu-Leu-Arg-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-Ser-Gly
In a preferred embodiment of the invention, the intranasal influenza vaccine consists of a mixture of the four influenza virus epitopes: hemagglutinin epitopes HA91-108 and HA307-319, and nucleoprotein epitopes NP335-350 and NP380-393, expressed individually in Salmonella flagellin. For non-Caucasian populations, other CTL epitopes can be used.
The present invention also relates to the use of a mixture of flagella containing at least four epitopes of influenza virus each expressed individually in Salmonella flagellin, as described above, for the preparation of a human synthetic influenza vaccine for intranasal administration.
The present invention further relates to a method for inducing a human immune response and conferring protection against influenza virus in humans, which comprises administering intranasally to human individuals a synthetic peptide-based influenza vaccine comprising a mixture of flagella, as described above.